Treatment of conditions related to shock

ABSTRACT

Techniques are disclosed for prevention or treatment of physiological shock by administering a specific therapeutic agent, which is able to use smaller volumes of reagent to achieve complete inhibition, than other previously described techniques.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. Ser. No. 15/010,058, filedJan. 29, 2016, now pending, which is a divisional of U.S. Ser. No.12/681,510, filed Aug. 9, 2010, now issued as U.S. Pat. No. 9,272,034,which is a U.S. national stage application under 35 U.S.C. 371 ofInternational application No. PCT/US2008/011529, filed Oct. 6, 2008,which claims priority to U.S. Provisional Patent Application Ser. No.60/977,587, filed Oct. 4, 2007, and to U.S. Provisional PatentApplication Ser. No. 60/980,430, filed Oct. 16, 2007, the contents ofall of which are hereby incorporated by reference in their entirety intothis disclosure.

GRANT INFORMATION

This invention was made with government support under Grant Nos.HL-10881, HL-067825 and HL-43026 awarded by National Institutes ofHealth. The United States government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to treatment of shock. In particular, thepresent invention relates to treatment of conditions related to shock.

Background Information

Shock is a life-threatening complication in situations associated withtrauma including burns, surgery, ischemia, sepsis, and other criticalcare applications. Shock is a broad term that describes a group ofcirculatory syndromes, all of which result in general cellular hypoxia.This leads to a depletion of the adenosine triphosphate (ATP), thefailure of the sodium-potassium pump, mitochondrial dysfunction, andultimately the release of a variety of toxic substances, includingsuperoxides. Superoxides are toxic to essentially all tissues. Theyreact with proteins and cause unfolding and are able to induce DNAdamage. Additionally, cellular activation in the circulation can bedetected by leukocytes or endothelial cells resulting in superoxideproduction, pseudopod projections, enzyme release, cytokine release, andexpression of membrane adhesion molecules. Cell activation fundamentallyalters the biomechanics of microvascular blood flow by a shift inrheological, adhesive, and cytotoxic cell properties. Eventually thesestress responses give rise to irreversible cardiovascular collapsebecause of their combined effects on the microcirculation.

There are few satisfactory drugs, treatment methods, or interventionsavailable for the prevention of conditions related to shock. Mostcurrently available methods for the treatment of such conditions relatedto shock deal with the symptoms, rather than the cause. For this reason,current clinical approaches are limited in their efficacy and can onlyprevent further damage from occurring.

Thus, there is a need in the art for a more effective treatment ofconditions related to shock. The treatment should be simple toadminister, effective and capable of aiding in emergency situations.

SUMMARY OF THE INVENTION

The present invention is a technique for treatment of conditions relatedto physiological shock by administering a more specific combination oftherapeutic agents, which is able to use smaller volumes of reagent toachieve complete inhibition, than other previously described methods,for example, that in U.S. Pat. No. 6,534,283, which is incorporated byreference herein in its entirety. The present invention is based upon anew hypothesis for the cause of shock and multi-organ failure:self-digestion through gut ischemic complications rather thanbacterial/endotoxin invasion.

The present invention dramatically reduces symptoms of multi-organfailure and mortality in septic shock associated with leakage of cecalmaterial into the peritoneum (e.g., cecal ligation shock). Furthermore,the present invention reduces symptoms of insulin resistance in shock(e.g., septic, hemorrhagic and cecal ligation shock). The methods weretested and verified in various animal studies as discussed below.

In experimental models, it was demonstrated that blockade of pancreaticenzymes in the lumen of the intestine in combination with treatmentagainst cytotoxicity in the peritoneum (blockade of digestive enzymes,binding of cytotoxic mediators and anti-bacterial treatment in theperitoneum) leads to a dramatic enhancement of survival rate in a modelof septic shock (cecal ligation model).

In experimental models, it has further been demonstrated that plasma ofanimals (such as rats) in shock produced by cecal ligation have plasmathat exhibits protease activity. The activity is sufficient to cleavethe binding domain of insulin on the insulin receptor alpha.Introduction of Futhane and Doxycycline attenuates the insulin receptorcleavage. It is expected that other symptoms of cell and organdysfunction (such as arterial vasospasm, immune suppression, enhancedpermeability, apoptosis, etc.) characteristic for shock will also beattenuated by this treatment.

Such findings lead to the present invention resulting in treatmenttechniques for prevention of multi-organ failure and mortality in septicshock associated with leaks from intestine during surgery, puncturedintestine, ruptured intestinal legions or appendix, or other any othersituation associated with leakage of intestinal material (e.g., cecal orfecal matter). Further, such treatments would lead to prevention of themetabolic syndromes in trauma patients and patients in the ICU.

In certain exemplary embodiments, the present invention is a method forprevention or treatment of physiological shock. The method includesadministering to a peritoneum of an individual a therapeutic dose of anycombination of one or more of: pancreatic digestive enzyme inhibitor,cytotoxic mediator inhibitor, and antibacterial agent.

A method according to the present invention blocks formation ofinflammatory mediators by pancreatic digestive enzymes in the intestinein septic shock and thereby reduces symptoms of multi-organ failure andsignificantly reduces mortality rate. It also serves to reduce morbidityand reduce post-operative complications, enhance recovery rate, andshorten hospital stays.

The treatment is administered into the lumen of the intestine to blockfully activated digestive enzymes and auto-digestion of the intestine.The treatment is highly effective to attenuate prolonged formation ofinflammation in septic shock, destruction of the intestinal epitheliallining, and reduces mortality. There is currently no comparabletreatment for septic shock.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes techniques for treatment of conditions relatedto shock. Various exemplary embodiments are presented to provide a broadspectrum of treatment available and application to such conditions.

As discussed above, the strategy for inhibiting gut enteral function hasbeen described in U.S. Pat. No. 6,534,283, which is incorporated byreference herein in its entirety. This patent describes the use ofprotease inhibition in the lumen of the gut in principle and morespecifically using specific commercially available protease inhibitors.The current strategy proposes numerous applications related topancreatic protease inhibitors.

In the present invention, treatment is administered into the lumen ofthe intestine in combination with a treatment of the peritoneal cavitythat can be administered after onset of shock.

In one series of experiments, the inventors discovered that delayedinhibition of digestive enzymes in the lumen of the intestine reducesinflammatory markers after shock. As a clinically relevant situation,the inventors examined the effectiveness of a delayed intestinalprotease inhibition during reperfusion after SMAO. Male Wistar rats wereexposed to superior mesenteric artery occlusion (SMAO) for 100 min andtreated by delayed intestinal lavage beginning 40 min after reperfusionwith either buffer or a reversible digestive protease inhibitor (FOY,0.37 mM). Arterial pressure during reperfusion was significantly lowerin shock animals compared with sham shock animals. SMAO and reperfusionwithout protease blockade caused the formation of leukocyte activationfactors in intestinal homogenates and in plasma, as well as intestinalinjury and also caused a significant increase in activated leukocytes invenules of cremaster muscle. In contrast, with digestive proteaseinhibition in the intestine, delayed lavage at 40 min after reperfusionled to a highly significant restoration of the initial blood pressurebefore shock, decreased formation of intestine-derived leukocyteactivation factors and intestinal injury. Delayed digestive enzymeblockade also caused lower leukocytes adhesion in post-capillary venulesand reduced cell death in the cremaster muscle microcirculation.Intestinal ischemia-induced endotoxemia was prevented by digestiveenzyme inhibition.

In summary, delayed intestinal protease inhibition serves to improveexperimental SMAO-induced shock by reducing intestinal injury, the levelof cell activation in plasma and in the microcirculation, and byrestoring the blood pressure.

Another series of experiments were performed to show that inhibition ofpancreatic digestive enzymes in the lumen of the intestine reduces theneed for resuscitation in hemorrhagic shock. The inventors examined theutility of intestinal lavage in a porcine model of hemorrhagic shock. Anobjective of this study was to determine the effect of digestiveprotease blockade in the lumen of the intestine during hemorrhagicshock. The animals (16 pigs) were subjected to a shock that mimicsclinical events. Pigs were bled 30 ml/kg over 30 minutes and maintainedat a mean arterial pressure of 30 mmHg for 60 minutes and shed blood wasthen used to maintain a pressure of 45 mmHg for three hours. Bothtreated pigs (6-amidino-2-naphthyl p-guanidobenzoate dimethane-sulfate(ANGD), 100 ml/kg of 0.37 mM in GOLYTELY, PEG-3350 and electrolytes fororal solution, via a duodenal catheter at 1 liter/hr directly into thelumen of the intestine) and controls (GOLYTELY, PEG-3350 andelectrolytes for oral solution, only) had significant reductions inprotein and protease levels in the duodenum during enteroclysis, howeveronly ANGD treated animals had persistent suppression of proteaseactivity in the intestinal lumen and in plasma throughout theexperiment. Pigs with blockade of digestive enzymes had a majorreduction of transfusion requirement of shed blood (18.1±4.5 ml/kgversus 30±0.43 ml/kg; p=0.002), a significantly lower level ofneutrophil activation than controls after resuscitation (31.1±3.3%versus 46.9±4.5% in controls, p=0.0002). Leukocyte infiltration into thelung was lower in treated than control animals (p=0.04) and the liverand small intestine showed less injury in treated animals. In summary, adigestive enzyme inhibitor given via enteroclysis significantly reducesleukocyte activation and transfusion requirements during resuscitationfrom hemorrhagic shock.

In another series of studies, the inventors show that blockage ofdigestive enzymes in the lumen of the intestine attenuates microvascularinflammation in peripheral organs. These experiments were designed toexamine whether inflammatory mediators generated in the intestine bydigestive enzymes are released early into the circulation and maycontribute to the severe systemic inflammatory response syndrome duringshock, a condition that involves the microcirculation in peripheralorgans. Intestinal ischemia and reperfusion-induced hypotension uponreperfusion was accompanied by a significant increase in the level ofneutrophil activating factors in the intestine and plasma. Duringreperfusion a significant increase in leukocyte-endothelium interactionsin post-capillary venules and parenchymal cell death are observed in thecremaster muscle in controls after SMAO. In contrast, intra-intestinalpancreatic protease inhibition (gabexate mesilate, 0.37 mM) results in astable blood pressure throughout the experiment. Cell activation andleukocyte-endothelial interactions, both in term of rolling and firmadhesion to the endothelium and cell death (as measured by propidiumiodine labeling) in the cremaster muscle, were almost completelyabolished after blockade with gabexate mesilate. In addition,ischemia-induced intestinal mucosal injury is attenuated with intestinalpancreatic protease inhibition. In conclusion, intestinal pancreaticprotease inhibition significantly attenuates intestinal ischemia-inducedshock by reducing the systemic inflammatory response syndrome.

In another series of experiments, the inventors show that digestiveenzyme blockade is protective against inflammation in shock if placedinside the lumen of the intestine and less by intravenousadministration. From a mechanistic point of view, an important featureis the fact that the significant protection rendered by the inhibitionof pancreatic digestive enzymes is only provided if the enzymes areblocked inside the lumen of the intestine. If the enzyme inhibitors areadministered directly into the circulation (ib., i.a., i.m.), less, andin some cases no protection is achieved, a feature that has beenconfirmed using different protease inhibitors (ANGD and aprotinin). Thisobservation supports the hypothesis that digestive enzymes in the lumenof the intestine—where they are fully activated and in highconcentrations—are the major enzymes in acute intestinal ischemia. Theyproduce inflammatory mediators that are carried towards the centralcirculation via the portal venous system, but also via the intestinallymphatics. Besides the portal venous circulation and the intestinallymphatics, the inventors showed that inflammatory mediators can also becarried directly across the intestinal wall into the peritoneal cavity,along a third major pathway.

In another study, the inventors show that digestive enzymes mediatemicrovascular inflammation in septic shock. Sepsis is accompanied bysevere inflammation whose mechanism remains uncertain. The inventorsexamined the possibility that pancreatic digestive enzymes may also beinvolved in inflammation in an experimental form of septic shock with alethal dose of endotoxin in the rat. Immediately after intravenousendotoxin administration, the small intestine was subjected tointra-luminal lavage with and without an inhibitor of pancreaticdigestive proteases (FOY, gabexate mesilate). After endotoxinadministration (4 mg/kg, gram-negative), control rats developedhypotension, tachycardia, hyperventilation and leukopenia. The intestineand plasma contained mediators that activated leukocytes. Theleukocyte-endothelial interaction within the cremaster musclemicrocirculation was enhanced. Endotoxin administration resulted inelevated IL-6 plasma levels and histological evidence indicates liverand intestinal injury. In contrast, blockade of pancreatic proteases inthe intestinal lumen significantly improved hemodynamic parameters andreduces all indices of inflammation in plasma as well as cell injury inperipheral skeletal muscle microcirculation. These experiments indicatethat inflammatory mediators derived from the intestine by digestiveproteases may be involved in the prolonged inflammatory response and maysustain symptoms of sepsis after an endotoxin challenge. A bolusadministration of endotoxin causes a transient inflammation response andelevated intestinal permeability. But the sustained inflammation thatleads to multi-organ failure in this situation is caused byauto-digestion due to escape of pancreatic digestive enzymes from thelumen of the intestine due to the elevated mucosal permeability.

A study of the long term survival after blockade of digestive enzymesprovided further support to the findings. In preparation for thisapplication the inventors carried out pilot studies in the rat with (a)hemorrhagic, (b) endotoxic and (c) cecal ligation shock, followed by anobservation period for two weeks until normal cage activities wererecorded in survivors. Without food restriction, in hemorrhagic shockthe mean blood pressure was reduced for two hours to 35 mmHg followed byreturn of all blood volume but no further resuscitation. The digestiveenzymes were blocked at 1 hour after hypotension by direct infusion ofANGD (0.37 mM, 15 ml) into the intestinal lumen after a temporaryexposure via a midline incision. In endotoxic shock, the rats receivedgram-negative endotoxin (5 mg/kg, ib.); the digestive enzymes wereblocked in the same way at 1 hour after endotoxin administration. Noother agent was administered. In cecal ligation shock both the digestiveenzymes in the lumen of the intestine as well as inside the peritoneumwere blocked with ANGD. Untreated controls in each model of shock hadhigh mortality (within less than 8 hours), while blockade of thedigestive enzymes ANGD in each shock model lead to a significantlyenhanced survival rate (Table I). In contrast to untreated controls, alltreated survivors returned after anesthesia within hours to normalactivity (walking, climbing, grooming, drinking, eating, bowelmovements) and within 3 days to normal weight gain. Furthermore,treatment with alternative serine protease inhibitors (CYCLOKAPRON,tranexamic acid; TRASYLOL, aprotinin) in cecal ligation shock gavesignificant survival rates (5/5 rats, P<0.0079; 4/5 rats, P<0.02,respectively).

TABLE I Long-Term Survival Following Shock With and WithoutIntra-Intestinal Enzyme Blockade* (A) Hemorrhagic (B) Endotoxic (C)Cecal Lig. Shock¹ Shock² Shock³ Non- Non- Non- Survivor SurvivorSurvivor Survivor Survivor Survivor Untreated 9  3 9  4 9 1 ANGD 2 10 110 1 9 Treated *number of rats ¹P < 0.01 ²P < 0.004 ³P < 0.001 byFisher's Exact Test

It was further shown that plasma of shock rats has protease activity andcauses cleavage of the extracellular domain of the insulin receptor,E-cadherin, and CAT-1. In all forms of shock there is consistentlyproteolytic activity in plasma. Therefore the inventors investigated theability of central venous plasma of rats in hemorrhagic shock (collectedat 4 hours) to cleave the extracellular domain of the insulin receptor aby using an antibody against the extracellular binding domain of insulincombined with membrane receptor density measurements. Exposure of normaldonor cells to plasma from shock rats, but not to plasma of controlrats, causes extensive cleavage of the insulin binding domain.Furthermore, this cleavage causes also a reduction of the glucosetransport into the cell cytoplasm. These results show that the plasmaenzymatic activity may be responsible for the development of insulinresistance typical for patients in shock. The activity can besignificantly blocked with ANGD (by more than 50%, results not shown),suggesting that proteases are a major component of this enzyme activity.There is also a significant cleavage of the extracellular domain of thetight junction protein E-cadherin in intestinal epithelium and CAT-1 inleukocytes.

Another recent study by the inventors, listed as item (9) below, andwhich is incorporated by reference herein in its entirety, showed thatpancreatic enzymes generate cytotoxic mediators in the intestine. And,thus, there exists a link between the permeability increase in theintestinal wall and the early stages of shock with formation ofinflammatory and cytotoxic factors. These factors may either be alreadypresent in form of digested food or may be created by action ofdigestive enzymes on interstitial structures after entry into theintestinal wall and may cause the intestinal necrosis observed in shock.We have shown that both individual serine proteases and fluid from thelumen of the intestine with endogenous proteases have the ability togenerate cytotoxicity from intestinal wall homogenates and that luminalfluid may also generate cytotoxicity from homogenized food. Thesefindings further support the hypothesis that lavage of the content ofthe small intestine with broad-spectrum inhibitors may be protective inshock, in line with experimental evidence. There is a need to identifythe actual biochemical structure of the cytotoxic factors and determinetheir mechanism of action.

In another follow up study, listed as item (10) below and incorporatedby reference herein in its entirety, the inventors sought to show thatthe intestine is a source of cytotoxic mediators in shock, and the roleof free fatty acids and degradation of lipid-binding proteins. In thisstudy, the inventors showed that using chloroform/methanol separation ofrat small intestine homogenates into lipid fractions and aqueous andsedimented protein fractions and measuring cell death caused by thosefractions, it was found that the cytotoxic factors are lipid in nature.Recombining the lipid fraction with protein fractions prevented celldeath, except when homogenates were protease digested. Using afluorescent substrate, the inventors found high levels of lipaseactivity in intestinal homogenates and cytotoxic levels of free fattyacids. Addition of albumin, a lipid binding protein, prevented celldeath, unless the albumin was previously digested with protease.Homogenization of intestinal wall in the presence of the lipaseinhibitor orlistat prevented cell death after protease digestion. Invivo, orlistat plus the protease inhibitor aprotinin, administered tothe intestinal lumen, significantly improved survival time compared withsaline in a splanchnic arterial occlusion model of shock. These resultsindicate that major cytotoxic mediators derived from an intestine underin vitro conditions are free fatty acids (FFAs). Breakdown of free fattyacid binding proteins by proteases causes release of free fatty acids toact as powerful cytotoxic mediators.

The discovery further includes clarification of the mechanism that leadsto insulin resistance. It is shown that one of the ways that the presentinvention works is due to enzymatic cleavage of the insulinbinding-domain, and introduction of proteases attenuates the process.

There is currently no generally accepted treatment algorithm or protocolfor treatment of insulin resistance in shock. Limited options includeinsulin administration.

In another recent study, listed as item (11) below and incorporated byreference herein in its entirety, the inventors showed that there is arelationship between proteinase activity and receptor cleavage and thatthere appears to be a mechanism for insulin resistance in thespontaneously hypertensive rat. The inventors hypothesized that enhancedproteolytic activity in the microcirculation of spontaneouslyhypertensive rats (SHRs) may be a pathophysiological mechanism causingcell membrane receptor cleavage and examined this for 2 differentreceptors. Immunohistochemistry of matrix-degrading metalloproteinases(matrix metalloproteinase (MMP)-9) protein showed enhanced levels in SHRmicrovessels, mast cells, and leukocytes compared with normotensiveWistar-Kyoto rats. In vivo microzymography shows cleavage by MMP-1 and-9 in SHRs that colocalizes with MMP-9 and is blocked by metalchelation. SHR plasma also has enhanced protease activity. The inventorsdemonstrated with an antibody against the extracellular domain that theinsulin receptor-α density is reduced in SHRs, in line with elevatedblood glucose levels and glycohemoglobin. There is also cleavage of thebinding domain of the leukocyte integrin receptor CD 18 in line withpreviously reported reduced leukocyte adhesion. Blockade of MMPs with abroad-acting inhibitor (doxycycline, 5.4 mg/kg per day) reduces proteaseactivity in plasma and microvessels; blocks the proteolytic cleavage ofthe insulin receptor, the reduced glucose transport; normalizes bloodglucose levels and glycohemoglobin levels; and reduces blood pressureand enhanced microvascular oxidative stress of SHRs. The results suggestthat elevated MMP activity leads to proteolytic cleavage of membranereceptors in the SHR, e.g., cleavage of the insulin receptor-bindingdomain associated with insulin resistance.

Further, there is currently no generally accepted treatment algorithm orprotocol for septic shock. There is an FDA approved treatment withactivated protein C (XIGRIS, drotrecogin alfa (activated), Eli Lilly),which gives a minor but confirmed survival benefit. However, even suchtreatment has been called into question as more recent trials could notconfirm the effectiveness of activated protein C.

Treatment of septic shock is based on supportive care by treating theunderlying infection (appropriate antibiotics within the first 4-8 hoursof presentation) and on restoring tissue perfusion with a combination offluid resuscitation (e.g., albumin, lactated or hypertonic saline) andvasopressor administration (e.g., noreinephrine).

In an exemplary embodiment, the present invention involves severalcomponents, which may be performed independently or in combination. Onecomponent of a treatment according to the present invention includesadministration of a pancreatic enzyme inhibitor directly into the lumenof the intestine (by oral administration, introduction via an esophagealcatheter, direct injection into the lumen of the intestine duringsurgery, etc.). The agents to be used individually or in combinationinclude but are not limited to: FUTHAN, nafamostat mesilate (0.37 mM);TRASYLOL, aprotinin (Aprotinin, Bayer) (1.4 mg/ml), serine proteaseinhibitor; CYKLOKAPRON, tranexamic acid (Pfizer) (1.4 mg/ml), serineprotease inhibitor; broad based MMP inhibitors (e.g., doxycycline);orlistat (5 to 50 mg/ml), lipase inhibitor; plus any other pancreaticenzyme inhibitor. The amount administered may be adjusted according tointestine size and enzyme levels to achieve complete blockade ofdigestive enzyme activity.

A second component of a treatment according to the present inventionincludes treatment of the peritoneum by a combination of threeprotective interventions: blockade of pancreatic digestive enzymes(serine proteases, lipases, as outlined in the first component describedabove); blockade of cytotoxic lipid derived mediators (e.g., free fattyacids) with free fatty acid binding proteins (e.g., albumin, andothers); antibacterial treatment against gram-positive and gram-negativebacteria that have entered into the peritoneal space (with antibiotictreatment, e.g., ciprofloxacin, metronidazole, imipenem and cilastatin,ticarcillin and clavulanate, cefuroxime). Further effectiveness of thetreatment is achieved by peritoneal/intraintestinal lavage incombination with the treatments listed above.

The administration of the serine proteases and MMPs with broad spectrumblockers, as outlined in the first component described above, may bealternatively or additionally performed through an intravenous (ib.)route.

The present invention may be used in numerous medical treatments,including but not limited to, treatment for prevention of multi-organfailure and mortality in septic shock. Any lipase inhibitor incombination with a pancreatic or leukocyte derived protease inhibitormay have utility to prevent inflammation in septic shock.

In one exemplary embodiment, which may be used for treatment forprevention of post-operational complications, including multi-organfailure, sepsis, morbidity, and mortality, pancreatic proteaseinhibition is initiated to reduce complications and hospital stay aftertrauma/surgery. Here, it has been shown that pancreatic enzymes in theintestine have the ability to generate powerful inflammatory mediatorsand that blockade of pancreatic enzymes in the lumen of the intestineattenuates inflammatory symptoms after different shock models.

In this embodiment, the present invention allows a reduction ininflammatory symptoms and complications (swelling, embolism formation,selected organ dysfunction, pulmonary embolism, incidence of stroke,patient mobility, morbidity, multi-organ failure, mortality) in any formof elective surgery/general anesthesia associated with elevated risks(such as prolonged surgery procedures, surgery with bypass requirements,surgery on patients with preconditions and risk factors, surgeryinvolving the intestine and pancreas). This results in a reduction inpost-surgical complications, enhance wound healing, reduce totalrecovery period, and reduce hospitalization requirements and time.

In elective surgery, pre-administration of a pancreatic enzyme inhibitormay be conducted directly into the lumen of the intestine (by oraladministration, introduction via an esophageal catheter, directinjection into the lumen of the intestine during surgery). The agents tobe used are individually or in combination: Futhane (0.1 mM); Trasylol(Aprotinin, Bayer) (1.4 mg/ml), serine protease inhibitor; cyclokapron(Pfizer) (1.4 mg/ml), serine protease inhibitor; Orlestat (5 to 50mg/ml), lipase inhibitor plus any other pancreatic enzyme inhibitor. Theamount administered is adjusted according to intestine size to achievecomplete blockade of digestive enzyme activity. The inhibitor isadministered prior to general anesthesia/surgery as pretreatment.

This is the first intervention against a major source of inflammation inmulti-organ failure associated with surgery/general anesthesia. Blockadeof digestive enzymes prior to general anesthesia may serve to preservebarrier properties of the intestinal mucosa, reduce inflammation in thecentral circulation, and consequently reduce recovery and wound healingperiods, post-surgical complications, hospital stays, etc.

A potentially useful application of the digestive enzyme inhibition aspre-treatment is for patients subjected to radiation or chemotherapeutictreatment. It could also work for radiation treatment under othercircumstances to reduce symptoms of multi-organ failure.

In another exemplary embodiment, the present invention provides a methodfor pancreatic protease inhibition in septic shock. There are many usesfor this embodiment, including but not limited to, treatment forprevention of multi-organ failure and mortality in septic shock. Suchtreatment works by blocking formation of inflammatory mediators bypancreatic digestive enzymes in the intestine in septic shock andthereby reducing symptoms of multi-organ failure and mortality.

The treatment is administered into the lumen of the intestine to blockfully activated digestive enzymes and auto-digestion of the intestine.The treatment is highly effective to attenuate prolonged formation ofinflammation in septic shock, destruction of the intestinal epitheliallining, and reduces mortality.

It is demonstrated that blockade of pancreatic enzymes in the lumen ofthe intestine attenuates inflammatory symptoms after administration of alethal dose of endotoxin (6 mg/kg). Experiments demonstrate reducedlong-term mortality in the same sepsis model.

Administration of a pancreatic enzyme inhibitor may be conducteddirectly into the lumen of the intestine (by oral administration,introduction via an esophageal catheter, direct injection into the lumenof the intestine during surgery). The agents to be used are individuallyor in combination: FUTHAN, nafamostat mesilate (0.1 mM); TRASYLOL,aprotinin (1.4 mg/ml), serine protease inhibitor; orlistat (5 to 50mg/ml), lipase inhibitor; plus any other pancreatic enzyme inhibitor.The amount administered is adjusted according to intestine size toachieve complete blockade of digestive enzyme activity.

In another exemplary embodiment, the present invention is used forpancreatic lipase inhibition to reduce mortality after shock. Thisembodiment is very useful for developing treatment for prevention ofmulti-organ failure and mortality in hemorrhagic shock, preventivetreatment to reduce the probability for development of multi-organfailure in elective surgery, long-term treatment to reduce production oflipid derived inflammatory mediators associated in chronic diseases. Itis also particularly useful because there does not appear to be anytreatment proposed to attenuate inflammation by blockade of lipaseactivity in the intestine in either acute or chronic inflammatoryconditions.

This embodiment is designed as an intervention to block the lipaseactivity in the lumen of the intestine and also in the generalcirculation in those cases in which lipase enters from the lumen of theintestine into the circulation. This prevents formation of lipid derivedinflammatory or cytotoxic mediators in shock and other inflammatorydiseases and attenuate multi-organ failure in shock and chronicinflammation in diseases like hypertension, diabetes, the metabolicsyndrome, cancers and in chronic degenerative diseases.

Recent evidence resulting in this invention suggests that a majorcomponent of inflammatory mediators from the intestine in shock causingmulti-organ failure and mortality (e.g., after surgery/generalanesthesia, trauma, chronic diseases and any other condition leadingmulti-organ failure) is derived from the action of pancreatic lipases(lipid splitting enzymes). Blockade of pancreatic lipase serves toreduce mortality during shock and reduce inflammation that leads tomulti-organ failure. Blockade of pancreatic lipase prior to generalanesthesia may serve to preserve barrier properties of the intestinalmucosa, reduce inflammation in the central circulation, and consequentlyreduce recovery and wound healing periods, post-surgical complications,hospital stays, etc.

The inventors have shown that the ischemic intestine produces a powerfulset of lipid derived cytotoxic mediators and that the blockade of lipasein the intestine under in-vitro conditions blocks the production oflipid-derived cytotoxic mediators.

In elective surgery, pre-administration of a pancreatic enzyme inhibitordirectly into the lumen of the intestine (by oral administration,introduction via an esophageal catheter, direct injection into the lumenof the intestine during surgery) may have a positive effect on recovery.The agents to be used are individually or in combination: orlistat (5 to50 mg/ml), lipase inhibitor; plus any other pancreatic enzyme inhibitor.The amount administered is adjusted according to intestine size andcontent to achieve complete blockade of digestive enzyme activity. Astreatment the inhibitor is administered after trauma or sepsisassociated with risk for shock and multi-organ failure. As pretreatmentthe inhibitor is administered prior to general anesthesia/surgery.

The above exemplary embodiments have shown various uses and techniquesfor decreasing certain conditions related to shock. Thus, as a whole,the present invention is based on data from animal studies that showdramatic reduction in life-threatening shock by inhibiting a body's ownaggressive digestive enzymes. This novel approach targets triggermechanisms in auto-digestion before it launches lethal inflammatorycascade.

Death from heart, lung and kidney failure during shock due to inadequateblood flow can be prevented by an unusual experimental treatment thatinhibits the aggressive enzymes that are produced in body to digestfood.

The invention provides evidence from recent animal studies that for thefirst time, studies showed that blockade of the digestive enzymes duringshock leads to long-term survival. The results show a dramatic reductionof mortality in hemorrhagic shock induced multi-organ failure. Thistreatment holds great promise for future clinical application,particularly in emergency rooms and before high-risk surgeries. When aperson is in shock, his or her life is on the line. The patient'ssurvival may be in jeopardy not just that day, but within an hourbecause healthy organs can fail and die in rapid succession.

An estimated 1 million cases of various types of shock are treatedannually in U.S. hospital emergency rooms. Shock is a serious medicalcondition with a fatality rate of approximately 29%. While the optimalmanagement of shock patients can improve survival rates, overall shockremains a condition with a high death rate.

Administering a drug to inhibit the body's digestive enzymes is arelatively new approach that was begun in the past decade. In 1998 afinding was made in laboratory studies on the body's inflammatorycascade and the factors that turn this normal tissue-healing biologicalprocess into a virulent, out of control firestorm against the body'snormal tissue.

The researchers then began animal studies. The present invention isbased on the latest research using rodent models of human hemorrhagicshock. Here it has been discovered that the sudden lowering of bloodpressure that occurs in people suffering from stroke can provoke thebody's digestive enzymes to break down the body's own intestinal tissueas if it were food. Such enzymes' abnormal actions may be defined as“auto-digestion.” Auto-digestion is dangerous because not only does itinjure healthy tissue but also contributes to multi-organ failure, whichcan be fatal.

The healthy cells of the animals' intestinal tissue react toauto-digestion by releasing a slew of substances that can be toxic tothe heart and other body organs. These substances, termed cytotoxicmediators, can reach these body organs via the blood stream. In theirlatest studies, shock was induced in 19 lab rodents, all of which werethen treated with therapies that mirror the emergency room care given tomany human patients who suffer shock, which typically occurs when bloodflow to the heart, lungs and other body organs is slowed as a result oftrauma, dehydration, heart attack or stroke.

A total of 10 of the 19 lab rodents in shock were also treated with theexperimental digestive enzyme inhibitor called ANGD. Eight of the tensurvived. However, only one of the nine “untreated” animals in shocksurvived. The other eight animals died from organ failure within 12hours. Although these “untreated” animals did not receive ANGD, theinhibitor, they were given basic shock care. The enzyme inhibitor ANGDdramatically improved the survival rate among the lab animals in whichshock had been induced.

In the pig studies, the scientists also are conducting experiments toidentify the time period when the experimental treatment will be themost effective in saving lives. The findings will be relevant to theemergency care of human patients in shock. Data indicate that theearlier the treatment occurs, the better the chances for survival.Current research indicates that the window of opportunity for thetreatment to be effective does not seem to be very narrow.

The discovery of the “auto-digestion” process and their positivefindings from the experimental treatment ANGD are based on NationalInstitutes of Health funded basic research to determine the origin ofthe inflammatory cascade that causes organ failure and death. Basically,inflammatory is the body's mechanism to repair, to heal tissue. But inshock, the inflammation never stops. It is out of control. Normally thebody senses when the inflammatory process has completed its job andbrings it to a halt.

There is little surprise that tissue can be severely damaged by theactions the body's digestive enzymes, which are secreted by the pancreasbut do not become activated until they arrive into the intestines.Digestive enzymes have to be very aggressive, and there has to be lotsof them, for the body to efficiently digest, to break down, the foodthat we eat. Normally the intestinal tissue is protected from theseenzymes by a layer of secreted mucus and by the tight packing of thecells in the intestinal wall. The enzymes are too big to defuse betweenthese cells under normal conditions.

The following references, some as cited above, are hereby incorporatedby reference herein in their entirety into this disclosure:

1. Schmid-Schonbein G W, Hugli T E. A New Hypothesis for MicrovascularInflammation in Shock and Multi-organ Failure: Self-Digestion byPancreatic Enzymes. Microcirculation. 2005; 12:71-82.

2. Doucet J J, Hoyt D B, Coimbra R, et al. Inhibition of enteral enzymesby enteroclysis with nafamostat mesilate reduces neutrophil activationand transfusion requirements after hemorrhagic shock. J Trauma. 2004;56:501-511.

3. Fitzal F, DeLano F A, Young C, Schmid-Schonbein G W. Improvement inearly symptoms of shock by delayed intestinal protease inhibition. ArchSurg. 2004; 139:1008-1016.

4. Deitch E A, Shi H P, Lu Q, et al. Serine proteases are involved inthe pathogenesis of trauma-hemorrhagic shock-induced gut and lunginjury. Shock. 2003; 19:452-456.

5. Shi H P, Liu Z J, Wen Y. Pancreatic enzymes in the gut contributingto lung injury after trauma/hemorrhagic shock. Chin J Traumatol. 2004;7:36-41.

6. Muhs B E, Patel S, Yee H, et al. Inhibition of matrixmetalloproteinases reduces local and distant organ injury followingexperimental acute pancreatitis. J Surg Res. 2003; 109:110-7.

7. Rosario H S, Waldo S W, Becker S A, et al. Pancreatic trypsinincreases matrix metalloproteinase-9 accumulation and activation duringacute intestinal ischemia-reperfusion in the rat. Am J Pathol. 2004;164:1707-16.

8. Fitzal F, DeLano F A, Young C, Rosario H S, Junger W G,Schmid-Schonbein G W. Pancreatic enzymes sustain systemic inflammationafter an initial endotoxin challenge. Surgery, 134:446-456, 2003.

9. Penn, A H, Hugli, T E, Schmid-Schonbein, G W. Pancreatic enzymesgenerate cytotoxic mediators in the intestine. Shock, Vol. 27, No. 3,pp. 296-304, 2007.

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The foregoing disclosure of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Many variations andmodifications of the embodiments described herein will be apparent toone of ordinary skill in the art in light of the above disclosure. Thescope of the invention is to be defined only by the claims appendedhereto, and by their equivalents.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention.

What is claimed is:
 1. A method for treating or preventing septic shockin an individual in need thereof, the method comprising administering toa peritoneum of the individual a therapeutic dose of: (i) a pancreaticdigestive enzyme inhibitor, (ii) a cytotoxic mediator inhibitor, (iii)an antibacterial agent, or (iv) a combination of two or more thereof. 2.The method of claim 1, further comprising administering to theindividual a therapeutic dose of an MMP inhibitor.
 3. The method ofclaim 1, wherein the MMP inhibitor is doxycycline.
 4. The method ofclaim 1, further comprising administering to an intestine of theindividual a therapeutic dose of: (i) a serine protease inhibitor, (ii)a lipase inhibitor, or (iii) a combination thereof.
 5. The method ofclaim 1, comprising administering the pancreatic digestive enzymeinhibitor.
 6. The method of claim 5, wherein the pancreatic digestiveenzyme inhibitor is nafamostat mesilate, aprotinin, tranexamic acid,orlistat, or a combination of two or more thereof.
 7. The method ofclaim 6, wherein the pancreatic digestive enzyme inhibitor is tranexamicacid.
 8. A method for treating or preventing septic shock in anindividual in need thereof, the method comprising administering into alumen of an intestine of the individual a therapeutic dose of a serineprotease inhibitor.
 9. The method of claim 8, wherein the serineprotease inhibitor is nafamostat mesilate, aprotinin, tranexamic acid,or 6-amidino-2-naphthyl p-guanidobenzoate dimethane-sulfate.
 10. Themethod of claim 9, wherein the serine protease inhibitor is tranexamicacid.
 11. The method of claim 8, wherein administering into a lumen ofan intestine comprises oral administration, introduction via anesophageal catheter, or direct injection into the lumen of theintestine.
 12. The method of claim 8, further comprising administeringto the peritoneal cavity of the individual a therapeutic dose of apancreatic digestive enzyme inhibitor.
 13. The method of claim 12,wherein the pancreatic digestive enzyme inhibitor is nafamostatmesilate, aprotinin, tranexamic acid, orlistat, or a combination of twoor more thereof.
 14. The method of claim 12, wherein the pancreaticdigestive enzyme inhibitor is a serine protease inhibitor.
 15. Themethod of claim 14, wherein the serine protease inhibitor is nafamostatmesilate, aprotinin, tranexamic acid, or 6-amidino-2-naphthylp-guanidobenzoate dimethane-sulfate.
 16. The method of claim 15, whereinthe serine protease inhibitor is tranexamic acid.
 17. A method fortreating or preventing septic shock in an individual in need thereof,the method comprising administering to the peritoneum of the individuala therapeutic dose of a serine protease inhibitor.
 18. The method ofclaim 17, wherein the serine protease inhibitor is nafamostat mesilate,aprotinin, tranexamic acid, or 6-amidino-2-naphthyl p-guanidobenzoatedimethane-sulfate.
 19. The method of claim 18, wherein the serineprotease inhibitor is tranexamic acid.